Nitrogen Metabolism and Nucleic Acid Metabolism V2.pdf

Module 2: Cell Nitrogen Metabolism and Nucleic Acid Metabolism Albert Tiotuyco, MD | August 28, 2024 | Asynchronous TABLE OF CONTENTS...

Module 2: Cell Nitrogen Metabolism and Nucleic Acid Metabolism Albert Tiotuyco, MD | August 28, 2024 | Asynchronous TABLE OF CONTENTS I. NITROGEN METABOLISM Learning Objectives A. Purpose of the Urea OVERVIEW Summary of Terms Cycle Amino Acids vs. Fats and Carbohydrates I. Nitrogen Metabolism B. Overview of the Urea →Fats and carbohydrates can be stored by the body for II. Amino Acids Catabolism Cycle later use A. Phase I C. Steps of the Urea Cycle →Ex. Fats as triglycerides or triacylglycerols in adipose B. Phase II D. Regulation of the Urea tissue. Glucose as glycogen in liver, skeletal muscle, III. Amino Acid Pool Cycle cardiac tissue. IV. Protein Turnover VIII. Purine and Pyrimidine →Cells can’t store amino acids for later use. V. Protein Degradation Synthesis →There is no protein that exists for this sole purpose. A. Ubiquitin-Proteasome A. Overview of Purine and Our cells can obtain amino acids in 3 ways: Pathway Pyrimidine Synthesis →De novo synthesis: synthesizing amino acids from B. Lysosomal Degradation B. Purine Synthesis scratch VI. Amino Acid Metabolism C. Pyrimidine Synthesis →Diet: Dietary intake and breakdown of protein (that can A. Overview of Amino Acid IX. Purine and Pyrimidine then form amino acids) Metabolism Catabolism →Breakdown and turnover of endogenous proteins that B. Fed State Amino Acid A. Purine Breakdown exists within cells within our body Metabolism B. Purine Salvage Pathway Endogenous proteins - proteins made inside the cell C. Fasted State Amino Acid C. Pyrimidine Breakdown II. AMINO ACID CATABOLISM Metabolism X. Summary & Key Points Any excess amino acids obtained from our diet that are not D. Breakdown of Proteins XI. Review Questions used or needed by our cells must be rapidly broken down. Through the Removal of XII. References Amino acid catabolism is divided into two phases. Amino Group Appendix A. PHASE I VII. Urea Cycle Freedom Space Removing α-amino group from amino acid LEARNING OBJECTIVES Involves transamination and oxidative deamination 1. Differentiate and enumerate the nutritionally essential vs. →Gives rise to ammonia (NH3) and α-keto acid the nutritionally non-essential amino acids →Some ammonia are secreted directly into the urine via 2. Describe the synthesis of the various nutritionally the kidneys non-essential amino acids →Majority of ammonia is transported to the liver where the 3. Explain the processes involved in the catabolism of urea cycle converts ammonia to urea, which can be proteins transported to kidneys → excretes urea via urine. 4. Describe the basic steps in the Urea Cycle B. PHASE II 5. Explain the importance of the Urea Cycle α-keto acid contains carbon skeleton which may be used 6. Describe the catabolism of the carbon skeletons of amino in this phase acids α-keto acid builds intermediate molecules of metabolic 7. Differentiate glucogenic from ketogenic amino acids pathways like the Krebs cycle 8. Describe certain disorders related to a dysfunctional urea May be used to help form glucose, fatty acids, ketone cycle and their medical consequences bodies → form ATP energy molecules 9. Describe how purines and pyrimidines are synthesized 10. Explain how uric acid is synthesized from purines 11. Describe the catabolism of pyrimidines SUMMARY OF TERMS Endogenous Proteins made inside the cell proteins Proteasome A large barrel-shaped complex intracellular proteases that function in regulated degradation of cellular proteins De novo synthesis Formation of nitrogenous bases from simple precursor molecules, rather than recycling pre-existing bases Salvage pathways Recycling of pre-existing purine and pyrimidine bases from normal cell turnover to synthesize nucleotides Gout A form of inflammatory arthritis caused by the deposition of monosodium urate crystals CELL 02.11 TG 8 | Arenas, Bauzon, Ferrer, Katipunan, Lavarias, Miraña, Rolloque, Rosello, Sanchez, See, Ty, Villanueva 1 of 14 CG 17 | Flores, Del Rosario, Guerrero, Jabagat, Lucero, Luna, Meris, Nangan, Pablico, Sablan, Valera  Figure 1. Urea Cycle as part of the essential pathways of energy metabolism III. AMINO ACID POOL Represents the collective mixture of amino acids that exists in the body that comes from the 3 processes: Figure 2. Amino Acid Pool →De novo synthesis →Dietary intake and breakdown of protein IV. PROTEIN TURNOVER →Breakdown and turnover of endogenous proteins) Once cells synthesize proteins, proteins do not exist On average, the amino acid pool is roughly about ~90 to forever, and must be broken down and reformed 100 grams The rate of protein turnover varies widely for individual The pool is depleted through: proteins (T02.12, 2028) →Synthesis of body protein Some cells have longer half-lives than others →Consumption of amino acids as precursors of essential →Example: Collagen is a very metabolically stable protein nitrogen-containing small molecules and its half life could be anywhere from months to years →Conversion of amino acids = long time before the cells can break down and reform it Can use amino acids to form new enzymes, proteins, and →In contrast, other proteins which are used very quickly other important biological molecules (e.g., transmitters, and have to be broken down (e.g. regulatory proteins) norepinephrine, dopamine, porphyrin, purines, pyrimidines immediately. to help form nucleic acid) Can take only minutes to hours to break down and At a stable state, it does not change (T02.12, 2028) reform →On average, cellular proteins have half lives somewhere between days to weeks. →Cellular proteins have an average of 300 to 400g daily turnover rate. V. PROTEIN DEGRADATION Ubiquitin-Proteasome Pathway: ATP-dependent ubiquitin (UB)-proteasome system of the cytosol Lysosomal Degradation: ATP-independent degradative enzyme system of the lysosomes UBIQUITIN-PROTEASOME PATHWAY Used predominantly in proteins synthesized inside and by the cell Energy-dependent; requires ATP hydrolysis Ubiquitin units are added to target protein that needs to be broken down through covalent attachment of ubiquitin (T02.11a, 2027) →These ubiquitin tagged targeted proteins then enter a large barrel-shaped structure called proteasome. Proteasome is a garbage disposal for proteins. When proteins enter this structure, they are broken down into CELL 02.11 Nitrogen Metabolism and Nucleic Acid Metabolism 2 of 14  amino acids, which will then be reused by the cell to build LYSOSOMAL DEGRADATION other proteins, enzymes, and biological molecules. Typically for proteins that are absorbed by the cell Nice! (TO2.12, 2028) (exogenous proteins) (TO2.12, 2028; T02.11a, 2027) → Specific Process (Abali et al., 2022): Ubiquitination of a Does NOT require ATP target protein occurs through isopeptide linkage of the →Lysosomes: structures in the cell that contain hydrolytic α-carboxyl group of the C-terminal glycine of Ub to enzymes, break down proteins absorbed by the cell the ε-amino group of a lysine by a three-step, Using acid hydrolases, nonselective degradation of: enzyme-catalyzed, ATP-dependent process (see Figure →Intracellular Proteins (autophagy) 3) →Extracellular Proteins (heterophagy) (T02.11a, 2027) ✦Enzyme 1: an activating enzyme that activates Ub ACTIVE RECALL BOX ✦Enzyme 2: a conjugating enzyme where activated Ub 1. How do the storage capabilities of fats and is transferred to after enzyme 1 carbohydrates differ from those of amino acids in the ✦Enzyme 3: a ligase that interacts with Enzyme 2-Ub body? and identifies the protein to be degraded a. Fats and carbohydrates are stored for later use, while → There is then an addition of four or more Ub molecules amino acids cannot be stored. to the target protein that generates a polyubiquitin b.Amino acids are stored for later use, while fats and chain carbohydrates cannot be stored. ✦Proteins tagged with Ub chains are then recognized c. Both fats and amino acids are stored for later use, by the proteasome while carbohydrates are not. → The proteasome unfolds, ubiquinates, and cuts the d. Carbohydrates and amino acids are stored for later target protein into fragments use, while fats are not. ✦The fragments are further degraded by cytosolic 2. Which of the following is NOT a way through which cells proteases to amino acids that will enter the amino can obtain amino acids? acid pool a. De novo synthesis ✦Ub is recycled b.Dietary intake c. Breakdown and turnover of endogenous proteins Proteins with specific chemical sequences are more likely d.Direct absorption from the environment to be broken down 3. What occurs during Phase I of amino acid catabolism? →Proteins with aspartate at the N-terminal are broken a. α-Keto acid is converted directly into glucose. down much more quickly, compared to proteins that do b.Ammonia is formed and then directly used to not have aspartate at the N-terminal end synthesize proteins. →Proteins which are rich in serine, proline, threonine and c. The α-amino group is removed, resulting in ammonia glutamate, are broken down more quickly than other and an α-keto acid. proteins d.Amino acids are immediately converted into fatty acids. 4. Which pathway for protein degradation requires ATP? a. Lysosomal Degradation b.Ubiquitin-Proteasome Pathway c. Both pathways require ATP d.Neither pathway requires ATP. 5. ________ proteins are proteins produced inside the cell. Answers: 1A, 2D, 3C, 4B, 5 Endogenous VI. AMINO ACID METABOLISM A. OVERVIEW Amino acid catabolism allows the production of ATP inside the cells Amino acid metabolism accounts for only 10-15% of the cell’s total energy production →Important for providing carbon backbones to support glucose synthesis (TO2.12, 2028; T02.10, 2025) →Makes smaller contributions compared to carbohydrate metabolism and fatty acid metabolism Two bodily states determine amino acid metabolism: FED vs FASTED state →Fed state Body’s state right after eating a meal ↑ insulin (in response to higher levels of blood Figure 3. The ubiquitin-proteasome degradation pathway of protein glucose) ↓ glucagon CELL 02.11 Nitrogen Metabolism and Nucleic Acid Metabolism 3 of 14  →Fasted state The liver is quite the centerpiece for metabolism Happens several hours after a meal In a fasted state, fatty acids are being released from ↓ insulin adipose tissues and being sent to the liver to be oxidized. ↑ glucagon (along with the other hormones) in Requires ATP response to lower levels of blood glucose →all the ATP are fueling the synthesis of glucose. →Insulin and glucagon regulate the bulk of metabolism in (AAs) FA → Liver → glucose our body →Ketones may be produced in extreme starvation state Amino acids are released from tissues (mostly muscle B. FED STATE AMINO ACID METABOLISM tissues) and are sent via the bloodstream to the liver The proteins we ingest in our food are broken down into Once the AAs have arrived in the liver, they can enter a amino acids inside our small intestine diverse array of metabolic pathways Once the Amino Acids are broken down in the small →Glucogenic amino acids may contribute to precursors of intestine, they travel via the bloodstream directly to the gluconeogenesis and help support the production of liver (just like glucose). glucose. Several things could happen afterwards: Those that become intermediates of the Krebs cycle →Liver sends amino acids to other tissues (e.g. amino may also contribute to the production of some ATP but, acids may be sent to muscles for their own protein in reality, only 10-15% of our total energy production is synthesis) (T02.12 2028) supplied by AAs. →Liver uses amino acids directly for protein synthesis FA still comprises the bulk of ATP production inside our →Liver converts excess amino acids to: body. Glucose The AAs are important for providing carbon backbones ○ Glycogen: ultimate storage form of this molecule in to support glucose synthesis. the liver →Ketogenic amino acids contribute to the synthesis of ○ Precursors: Pyruvate, Oxaloacetate (OAA) acetyl-CoA, and subsequently, ketones − OAA is in equilibrium with a lot of the Ketones synthesis preserves the degradation of intermediates of the Krebs cycle protein in the muscle until a more sustainable energy ○ Conversion of Glucogenic amino acids source is found. Thus, the acetyl-CoA that contributes Fatty acids to ketone synthesis largely comes from fatty acids ○ Triacylglycerides in the adipose tissue: ultimate (T02.12, 2028) storage form of fatty acids. ○ Precursor: Acetyl-CoA − Acetyl-CoA is in equilibrium with acetoacetyl-CoA ○ Conversion of Ketogenic amino acids Carbon backbone of amino acids can be interconverted and metabolized into the precursor molecules of glucose and fatty acids listed above (TO2.12, 2028) Nice! Two Types of Amino Acids → Essential VS Non-essential: ✦Essential amino acids: cannot be synthesized by the Figure 4. Summary of Amino Acid Metabolism (Khan Academy Medicine, 2024) body and are instead obtained from dietary sources ✦Non-essential amino acids: can be synthesized in the D. AMINO ACID STRUCTURE AND CATABOLISM body → Glucogenic VS Ketogenic: ✦Glucogenic: The carbon backbone feeds into the precursor molecules for glucose synthesis (e.g. pyruvate, oxaloacetate, and Krebs cycle intermediates) ✦Ketogenic: The carbon backbone feeds into the precursor molecules for fatty acid synthesis. (e.g. acetyl CoA, acetoacetyl CoA) − Lysine and leucine: exclusively ketogenic amino acids → fatty acid synthesis ✦Some amino acids may contribute to both pathways. Figure 5. Structures of Amino Acids Remember who is essential: PVT TIM HALL (T02.10, 2025) ✦Phenylalanine-Valine-Tyrosine Basic Structure of Amino Acids ✦Tryptophan-Isoleucine-Methionine →The presence of nitrogen in the amine group - unique to ✦Histidine-Arginine-Leucine-Lysine the breakdown of proteins →The amine group does not contribute in any way to the C. FASTED STATE AMINO ACID METABOLISM production of the precursor molecules stated earlier in CELL 02.11 Nitrogen Metabolism and Nucleic Acid Metabolism 4 of 14  regards to the breakdown of AAs; only the Carbon Answers: 1B, 2C, 3F, 4B, 5T backbone (enclosed in the square brackets in Figure 5). TRANSAMINATION VII. UREA CYCLE Occurs predominantly in the hepatocytes of our liver cells First step in the catabolism of amino acids Also occurs in our kidneys to a lesser extent The amine group is transferred to another molecule for A. PURPOSE eventual excretion by the body → frees up the carbon backbone to contribute to the rest of the metabolic Method of transforming toxic by-product of amino acid pathways metabolism, Ammonia (NH3), into a less toxic form, urea. Freed carbon backbone is now called an α-keto acid →Urea - major disposal form of amino groups derived from →Its name was derived from its structure: amino acids and accounts for ~90% of the nitrogen α: alpha carbon relative to the carboxylate ion containing components of urine (Abali et. al., 2020) Keto: ketone Urea is mobilized and transported to the kidneys, where it Acid: attached to a carboxylic acid functional group can then be excreted via urine. α-ketoglutarate B. OVERVIEW The common molecule that accepts the amine group from the amino acid is called α-ketoglutarate →An intermediate in Krebs cycle →When it accepts the amine group in this process, it becomes a molecule of glutamate →Once glutamate reaches the liver, it donates the amine group in the form of ammonia (NH3) which is in equilibrium with ammonium (NH4+) →NH3 enters the urea cycle inside of the liver where it will be converted into a molecule of urea which will then be excreted in the urine. This process shows how our body →effectively use the carbon backbone of these AAs →detoxifies the nitrogen-containing amine compound Ammonia is excreted because it is toxic at high levels. Figure 7. Urea cycle overview (Abali et al., 2022) Steps 1-2 occur in the matrix of the mitochondria of the liver cell (hepatocytes). Steps 3-5 take place in the cytoplasm. Step 1: Carbamoyl Phosphate Formation →Extra ammonium from amino acid metabolism is combined with CO2 from bicarbonate Figure 6. Transamination Step (Khan Academy Medicine, 2024) →2 ATP is utilized to form high energy molecule, I’m ACTIVE RECALL BOX Carbamoyl Phosphate 1. In the FED state, which of the following hormonal Step 2: Ornithine and Carbamoyl Phosphate combine to changes occurs? form Citrulline a. Increased glucagon and decreased insulin →Ornithine moves into the matrix to combine with b.Increased insulin and decreased glucagon Carbamoyl Phosphate to make Citrulline. c. Decreased insulin and decreased glucagon →Both are amino acids not among the 20 amino acids for d.Decreased insulin and increased glucagon protein synthesis. 2. What is the common molecule that accepts the amine Step 3: Citrulline turns into Argininosuccinate group during transamination? →Citrulline is combined with Aspartate to make a. Pyruvate Argininosuccinate b.Acetyl-CoA →1 ATP is used to make, Hydrolyzation of PPi occurs, using c. α-Ketoglutarate 1 more ATP d.Oxaloacetate Step 4: Argininosuccinate is broken down into Fumarate 3. T/F: Glucogenic amino acids contribute to the synthesis and Arginine of ketones and fatty acids. →Carbon skeleton of Aspartate will become Fumarate 4.Which amino acids are exclusively ketogenic? Fumarate is a bridge between gluconeogenesis and a. Phenylalanine and Valine the urea cycle. b.Lysine and Leucine →Second amino group from Aspartate ends up on Arginine c. Isoleucine and Methionine Step 5: Hydrolyzation of Arginine to Urea d.Histidine and Arginine →One molecule of H2O is added to Arginine to remove 5. T/F: During transamination, the α-amino group is Urea and produce Ornithine, which goes through the transferred to α-ketoglutarate to form glutamate. cycle back into the matrix. →Composition of urea: CELL 02.11 Nitrogen Metabolism and Nucleic Acid Metabolism 5 of 14  Carbon from CO2 (as bicarbonate) (of step 1) Nitrogen from Ammonium (of step 1) Other nitrogen from Aspartate (of step 3) Oxygen from H2O (of step 5) Figure 10. Bicarbonate to Carboxyphosphate (ANDREY K, 2016) The ammonia group would attach to the carboxyphosphate Figure 8. Structure of Urea to form Carbamic Acid. Note: Glutamate is the immediate precursor of both Note: Ammonia(NH3) is in equilibrium with ammonia (through oxidative deamination by GDH) and ammonium(NH4+) aspartate nitrogen (through transamination of oxaloacetate by AST) (Abali et al., 2023) Figure 11. Carboxyphosphate to Carbamic Acid (ANDREY K, 2016) The ammonia group would release the orthophosphate (PO43-) group from Carbamic Acid, which then reacts with ATP to form Carbamoyl Phosphate. →Carbamoyl Phosphate has high transfer potential due to the presence of the anhydride bond making it easy to react to Ornithine in the next step. Figure 12. Carbamic Acid to Carbamoyl Phosphate (ANDREY K, 2016) Synthesis of Citrulline →Enzyme: Ornithine Transcarbamoylase Figure 9. Flow of nitrogen from amino acids to urea (Abali et al., 2022) →Location: Mitochondrial matrix Net reaction: →Transfer the Carbonyl group from Carbamoyl phosphate CO2 + NH4 + Aspartate + 2H2O + 3ATP → to Ornithine, forming Citrulline, which is then transported Urea + 2ADP + AMP + 2Pi + PPi + Fumarate out into the cytosol through an ornithine-citrulline →The synthesis of urea is irreversible, with a large transporter (i.e. antiporter) (Abali et al., 2022) negative ΔG, due to the cleavage of many high-energy phosphate bonds (Abali et al., 2022) C. STEPS OF THE UREA CYCLE Formation of Carbamoyl Phosphate →Enzyme: Carbamoyl Phosphate Synthetase Activated by N-acetylglutamate →Location: Mitochondrial matrix Figure 13. Synthesis of Citrulline (ANDREY K, 2016) →CO2 is from Bicarbonate, which is phosphorylated into Synthesis of Argininosuccinate carboxyphosphate. →Enzyme: Argininosuccinate synthetase ATP is utilized to increase Bicarbonate’s energy to →Location: Cytoplasm attach to the ammonia group →Addition of 2nd Amino group from Aspartate to Citrulline, using 1 ATP. →ATP is hydrolyzed forming AMP and Pyrophosphate(PPi). Pyrophosphate is unstable and will be hydrolyzed into another ATP molecule (2 equivalent ATP molecules used) CELL 02.11 Nitrogen Metabolism and Nucleic Acid Metabolism 6 of 14  →Energy is used to form a bond between the Nitrogen from Aspartate and the Carbon from Citrulline, releasing oxygen. →Positive charge could be seen delocalized between 3 Nitrogen atoms of the product. Figure 17. Formation and degradation of N-acetylglutamate (Abali et al., 2022) Other regulators: →Substrate availability for short-term regulation →Enzyme induction for long-term regulation Figure 14. Citrulline to Argininosuccinate (ANDREY K, 2016) VIII. PURINE AND PYRIMIDINE SYNTHESIS Cleavage of Argininosuccinate into Fumarate and Purine and pyrimidine synthesis occurs in the cytosol Arginine 5-Phosphoribosyl-1-Pyrophosphate (PRPP) →Enzyme: Argininosuccinase →An important precursor in the synthesis of purine and →Location: Cytoplasm pyrimidine nucleotides and some amino acids →Argininoosuccinase removes the carbon skeleton from →Made from Ribose-5-Phosphate (R5P) Argininosuccinate to make Fumarate and Arginine. →Provides the phosphoribose unit for BOTH purine and →Fumarate is an important bridge between pyrimidine nucleotides gluconeogenesis and the urea cycle. →Phosphoribose unit includes pentose sugar and phosphate portion for the nucleotides Ribose-5-Phosphate (R5P) →A product of the pentose phosphate pathway (PPP) →R5P + ATP → AMP + PRPP →Catalyzed by ribose phosphate pyrophosphokinase also known as PRPP synthetase →Pyrophosphate from ATP is added to R5P Figure 15. Breakdown of Argininosuccinate to Fumarate and Arginine (ANDREY K, 2016) Production of Urea →Enzyme: Arginase This enzyme is virtually exclusive to the liver, thus only the liver can synthesize urea. →Location: cytoplasm →H2O is introduced to hydrolyze Arginine to form Ornithine and Urea. →Urea goes into the bloodstream, then to the kidney to be Figure 18. PRPP Structure removed as urine. Pyrophosphate comes from ATP, Phosphoribose comes Urea contains 2 nitrogen groups from Aspartate and from R5P Ammonium. A. PURINE SYNTHESIS OVERVIEW →Ornithine is recycled back into the Urea cycle. PRPP → Inosine -5’- Monophosphate (IMP) Inosine -5’- Monophosphate (IMP) →Precursor for purine nucleotides (Adenosine and Guanosine) →Can be used to create either GMP or AMP →Both GMP and AMP pathways require energy Figure 16. Arginine to Ornithine (ANDREY K, 2016) D. REGULATION OF THE UREA CYCLE Rate-limiting step: Carbamoyl Phosphate Synthetase (Step 1) →Requires N-acetylglutamate, which is synthesized from acetyl CoA and glutamate by N-acetylglutamate synthase →Arginine activates the N-acetylglutamate synthase Figure 18. IMP GMP and AMP Pathways ATP → most common energy currency in the cell GTP → also used as energy; produced in the TCA cycle ATP is utilized to make GTP GTP is utilized to make ATP Purine synthesis is regulated tightly CELL 02.11 Nitrogen Metabolism and Nucleic Acid Metabolism 7 of 14  →Prevents the waste of energy and nitrogen →Uric acid (purine waste product) is harmful Too much uric acid production leads to its deposition in joints leading to gout B. PYRIMIDINE SYNTHESIS Step 1: Production of Carbamoyl Phosphate →HCO3- + (Gln, Q) + 2ATP + 2H2O → Carbamoyl Phosphate + (Glu, G) + 2ADP + Pi →Enzyme: carbamoyl phosphate synthetase II (CPS II) Step 2: Synthesis of N-carbamoyl-L Aspartate →Removal of PI from carbamoyl phosphate →Addition of carbamoyl to L-aspartate →Enzyme: Aspartate Transcarbamoylase (ATCase) Step 3: Synthesis of Dihydroorotate Figure 19. Pyrimidine Breakdown Pathway →Removal of H2O from N-carbamoyl-L Aspartate →Creation of the pyrimidine ring Cytosine and Thymine are part of the DNA. →Enzyme: Dihydroorotase Step 1: When DNA is degraded, cytosine and thymine is Step 4: Synthesis of Orotate released →Oxidation of dihydroorotate Step 2: Cytosine is processed to uracil through a →Enzyme: Dihydroorotate dehydrogenase deamination reaction →Only enzyme in the inner mitochondrial membrane →Both uracil and thymine can be salvaged and repurposed Step 5: Synthesis of Orotidine Monophosphate (OMP) in the Pyrimidine Salvage Pathway →Release of Pyrophosphate (PPI) Step 3: Uracil and Thymine will both be acted on by →Addition of PRPP provides phosphoribose unit from R5P enzyme dihydropyrimidine dehydrogenase to produce →Irreversible dihydrouracil and dihydrothymine, respectively →Enzyme: Orotate phosphoribosyltransferase →This step requires NADPH Step 6: Decarboxylation of OMP to Uridine →Rate Limiting Step of Pyrimidine Breakdown Monophosphate (UMP) Step 4: Dihydrouracil is converted to →Enzyme: Orotidylate decarboxylase (OMP N-carbamoyl-B-Alanine and Dihydrothymine into decarboxylase) N-carbamoyl-B-Aminoisobutyrate →2 ATP added to UMP → UTP Step 5: Beta-ureidopropionase processes the N-carbamoyl Step 7: Amination of UTP to Cytidine Triphosphate (CTP) form of the two products into B-Alanine (uracil) and →Gln, Q added to UTP → CTP B-Aminoisobutyrate (Thymine). →Enzyme: CTP synthetase Step 6: These products will be processed further into Differences from purine synthesis: Acetyl CoA (B-Alanine) and Succinyl CoA →Ring is built (B-Aminoisobutyrate). →Towards the end, PRPP adds the phosphoribose unit Both of these products will be used in the TCA cycle. Some of the byproducts are Ammonia, CO2 ACTIVE RECALL BOX Ammonia is toxic so the body needs to get rid of it through 1. What enzyme adds carabamoyl to L-aspartate to form the urea cycle. N-carbamoyl-L Aspartate? 2. Where does purine and pyrimidine synthesis occur? B. PURINE BREAKDOWN a. Mitochondrial matrix b. Nucleus c. Mitochondria inner membrane d. Cytosol 3. T/F: Inosine -5’- Monophosphate (IMP) is converted to PRPP to form either AMP or GMP 4. T/F: Ribose-5-Phosphate (R5P) provides the Figure 20. Purine Nitrogenous Bases phosphoribose unit for purine nucleotides only Purine Breakdown Product - Uric Acid Answers: 1 Aspartate Transcarbamoylase (ATCase), 2D, →Toxic molecule so the body has to get rid of it and 3F, 4F excrete it properly →Daily Production: 600mg/L IX. PURINE AND PYRIMIDINE CATABOLISM →Low Plasma Solubility: 70mg/L A. PYRIMIDINE BREAKDOWN Figure 21. Uric Acid Pathway CELL 02.11 Nitrogen Metabolism and Nucleic Acid Metabolism 8 of 14  Uric acid enters the blood and becomes sodium urate. Purine Salvage Pathway Then, it goes to the kidney and is excreted as urine. This process is critical to get rid of uric acid from the blood. Purine Breakdown Pathway Figure 19. Purine Salvage Pathway Hypoxanthine and Guanine can be salvaged from this pathway Both salvaging requires PRPP Enzyme: Hypoxanthine-Guanine Phosphoribosyltransferase (HGPRT) Figure 18. Purine Breakdown Pathway →Hypoxanthine → IMP →Guanine → GMP Step 1: Starts with AMP, which can be processed to IMP can be processed into GMP or into AMP in the purine become adenosine and IMP by enzyme AMP deaminase synthesis pathway →NH4+ is released from this reaction HGPRT Deficiency → Lesch-Nyhan Syndrome Step 2: Adenosine can be processed to Inosine by enzyme X-Linked Adenosine Deaminase 1/380,000 live births →NH4+ is released Decreased Salvage -> Increased Catabolism -> Increased Step 3: IMP can be reprocessed into Inosine Uric Acid (Hyperuricemia) Step 4: Inosine is converted to Hypoxanthine by enzyme Increased PRPP -> Increased Uric Acid Production Purine Nucleoside Phosphorylase Gout = Deposition of Sodium Urate Crystals into Tissue →Releases Ribose-1-phosphate →Because sodium urate or uric acid has a very low Step 5: Hypoxanthine can be salvaged in the Purine solubility plasma (70 mg/L). So anything more than that Salvage Pathway or it can be further metabolized into will cause a precipitating effect and you can get them Xanthine by the enzyme Xanthine Oxidase lodged in tissues, leading to symptoms of gout. →H2O2 is produced Step 6: Xanthine can be further processed by Xanthine Nice! Oxidase into Uric Acid Nucleosides →H2O2 is produced → Produced by the addition of a pentose sugar to a base →Allopurinol - drug used for gout is an inhibitor of the through an N-glycosidic bond enzyme Xanthine Oxidase. This inhibits hypoxanthine → Ribonucleoside - produced if the pentose sugar is from being metabolized to xanthine, and eventually uric ribose acid. ✦Adenosine (A) ✦Guanosine (G) C. PURINE SALVAGE ✦Cytidine (C) GMP can be processed to Guanosine ✦Uridine (U) Guanosine is converted to Guanine using Purine → Deoxyribosucloside - produced if the pentose sugar is Nucleoside Phosphorylase 2-deoxyribose →Ribose-1-phosphate is released ✦deoxyadenosine (A) After multiple steps, Guanine can either be salvaged by ✦deoxyguanosine (G) Purine Salvage Pathway or be further metabolized by ✦deoxycytidine (C) enzyme Guanase to form Xanthine ✦deoxythymidine (T), or simply thymidine →NH4+ is released → The carbon and nitrogen atoms in the rings of the base →Xanthine will be acted upon by Xanthine Oxidase to form and sugar are numbered separately. Uric Acid Both GMP and AMP lead to the production of uric acid This pathway is critical for the proper metabolism of ACTIVE RECALL BOX purines and if there’s a problem with this pathway, dire 1. What are the three main components of a nucleotide? effects can occur such as: 2. What are the two ways in which purine and pyrimidine →Deficiency in Adenosine Deaminase (ADA) bases can be synthesized in the cell? Causes 15% of Severe Combined Immunodeficiency 3. What is the difference between uracil and thymine? (SCID) Answers: 1 Pentose monosaccharide, nitrogenous base, There are problems in this pathway that are critical to phosphate group/s, 2 De novo synthesis and salvage proper body health and homeostasis. pathways, 3 Thymine has a methyl group (Figure 20) CELL 02.11 Nitrogen Metabolism and Nucleic Acid Metabolism 9 of 14  A. RATIONALE TO ANSWERS OF REVIEW QUESTIONS 1. [T] - Transamination and oxidative deamination remove amino groups, producing ammonia (NH₃) and α-keto acids. 2. [F] - Pyrophosphate in PRPP comes from ATP 3. [B] - Tyrosine, phenylalanine 4. [A] - Dihydropyrimidine dehydrogenase 5. [C] - Maple syrup urine disease 6. [F] - Increased PRPP formation will lead to increased uric acid formation XII. REFERENCES REQUIRED RESOURCES Figure 20. Structures of thymine and uracil. Abali, E.E., Cline, S.D., Franklin, D,S. & Viselli, S.M. (2022). Lippincott's Illustrated Reviews: Biochemistry (8th ed.). Philadelphia, PA: Wolters Kluwer Health/Lippincott Williams X. SUMMARY & KEY POINTS & Wilkins. Amino acids are not stored by the body. Thus, amino AK Lectures. (2016). The urea cycle. [YouTube Video]. acids must be obtained from diet, synthesized de novo, or https://www.youtube.com/watch?v=VtDtG58ETLQ produced from degradation of protein. AK Lectures. (2019, August 6). Introduction to nitrogen Amino acid catabolism begins with the removal of metabolism. [YouTube Video]. alpha-amino groups by transamination and oxidative https://youtu.be/MUlvdoMDfG4 JJ Medicine. (2017a, April deamination, forming ammonia and alpha-keto acids. 19). Purine and pyrimidine catabolism pathway - nucleotide Free ammonia is excreted in the urine or used in urea breakdown - biochemistry lesson. [YouTube Video]. synthesis (urea cycle). https://youtu.be/oBMKSFGj_2E?feature=shared Carbon skeletons of the alpha-keto acids are converted to JJ Medicine. (2017, April 18). Pyrimidine Synthesis and intermediates of several metabolic pathways. Salvage Pathway [YouTube Video]. The committed step in purine synthesis uses PRPP. https://youtu.be/mjFDukNpVE4 The first and regulated step in pyrimidine synthesis uses Khan Academy Medicine. (2014). Overview of Amino Acid carbamoyl phosphate synthetase II to produce carbamoyl Metabolism. [YouTube Video]. phosphate. This is activated by PRPP and inhibited by https://www.youtube.com/watch?v=l0V-Xmps1mE UTP. Moof University. (2014). Purine and pyrimidine nucleotide XI. REVIEW QUESTIONS biosynthesis. [YouTube Video]. https://www.youtube.com/watch?v=pYhabLiXy4U #1 T/F: Phase I of amino acid catabolism involved transamination and oxidative deamination leads to the SUPPLEMENTARY RESOURCES production of ammonia (NH3) and α-keto acid. ASMPH Batch 2028. [02.12] Nitrogen Metabolism and True or False? Nucleic Acid Metabolism V2.pdf #2 T/F: The pyrophosphate in PRPP comes from R5P. True or False? #3: Which of the following amino acids form fumarate? A. Methionine, valine, isoleucine, threonine B. Tyrosine, phenylalanine C. Glutamine, proline, arginine histidine D. Asparagine #4: What is the enzyme involved in the rate-limiting step of pyrimidine breakdown? A. Dihydropyrimidine dehydrogenase B. Dihydroorotase C. Uridine phosphorylase D. Thymidine kinase #5: Which of the following disorders is autosomal recessive and is caused by partial or complete deficiency in BCKD? A. Phenylketonuria B. Albinism C. Maple Syrup Urine Disease D. Homocystinuria #6 T/F: Increased PRPP formation in the purine salvage pathway will lead to decreased uric acid formation. True or False? CELL 02.11 Nitrogen Metabolism and Nucleic Acid Metabolism 10 of 14  Inosine Monophosphate Synthesis APPENDIX 9 more steps occur leading to the synthesis of inosine monophosphate (IMP) (DETAILED) NUCLEOTIDE METABOLISM →IMP is the parent purine nucleotide for AMP and GMP OVERVIEW synthesis 4 steps require ATP as an energy source Purine and pyrimidine bases in nucleotides can be 2 steps require N10-formyl-THF as a one-carbon donor synthesized in two ways: →De novo synthesis - formation of nitrogenous bases AMP and GMP Synthesis from simple precursor molecules, rather than recycling IMP is converted to either AMP or GMP in 2 steps. pre-existing bases →Requires energy and nitrogen →Salvage pathways - recycling of pre-existing purine and →The first reaction in each pathway is inhibited by the end pyrimidine bases from normal cell turnover to synthesize product of that pathway AMP synthesis requires GTP as the energy source and nucleotides aspartate as a nitrogen source. NUCLEOTIDE SYNTHESIS GMP synthesis requires ATP as the energy source and glutamine as a nitrogen source. Purine Nucleotide Synthesis Nucleoside di- and triphosphate Synthesis Liver: the primary site of purine ring synthesis Nucleoside monophosphate kinases synthesize nucleoside A series of reactions add carbons and nitrogens from diphosphates from corresponding nucleoside monophosphates various compounds to a preformed ribose 5-phosphate →The enzymes are base-specific Atoms of purine rings come from various compounds such →These enzymes do not discriminate between ribose or as: deoxyribose in the substrate → Carbon dioxide (CO2) The transferred phosphate is from ATP → Amino acids (aspartate, glycine, and glutamine) Adenylate kinase is particularly active in the liver and muscle →N10-formyltetrahydrofolate (N10-formyl-THF) (sites of high ATP energy turnover) 5-Phosphoribosyl-1-pyrophosphate (PRPP) synthesis Nucleoside diphosphate kinase interconverts nucleoside diphosphates and triphosphates An activated pentose that participates in the synthesis and →Broad substrate specificity salvage of purines and pyrimidines Synthesized from ATP and ribose 5-phosphate, catalyzed Purine Salvage Pathway by PRPP synthetase Purines from normal turnover of nucleic acids or from diet →Activated by inorganic phosphate and inhibited by purine can be converted to nucleoside triphosphates nucleotides (end-product inhibition) Adenosine is the sole purine nucleoside to be salvaged →Transfers the phosphate moiety of ATP to carbon 1 of →Phosphorylated to AMP by adenosine kinase ribose 5-phosphate, forming PRPP Two enzymes are involved The sugar moiety of PRPP is ribose →Adenine phosphoribosyltransferase (APRT) →End products of de novo purine synthesis = →X-linked hypoxanthine-guanine ribonucleotides phosphoribosyltransferase (HGPRT) →When deoxyribonucleotides are needed for DNA Both enzymes use PRPP as the source of the ribose synthesis, the ribose sugar moiety is reduced 5-phosphate group Reactions are irreversible →Pyrophosphate is released and hydrolyzed by pyrophosphatase ACTIVE RECALL BOX 1. T/F: PRPP synthetase is activated by inorganic Figure 21. PRPP synthesis phosphate 2. T/F: AMP synthesis requires GTP as the energy source 5-Phosphoribosylamine Synthesis and aspartate as the nitrogen source. Synthesized from PRPP and glutamine 3. 5-Phosphoribosylamine is synthesized from what The amide group of glutamine replaces the pyrophosphate substrates? group attached to carbon 1 of PRPP, catalyzed by glutamine: 4.What is the committed step in purine nucleotide phosphoribosylpyrophosphate (GPAT) biosynthesis? →Committed step in purine nucleotide biosynthesis 5. T/F: Nucleoside monophosphate kinases have broad GPAT is inhibited by the end-products of the pathway: AMP substrate specificity and GMP Answers: 1T, 2T, 3 PRPP and glutamine, 4 Replacement of The intracellular concentration of PRPP controls the rate of the reaction the pyrophosphate group in carbon 1 of PRPP by the amide →PRPP concentration is normally far below the KM for GPAT, group of glutamine as catalyzed by GPAT, 5F - They are so any small change in PRPP concentration changes base-specific reaction rate largely. CELL 02.11 Nitrogen Metabolism and Nucleic Acid Metabolism 11 of 14  DEOXYRIBONUCLEOTIDE SYNTHESIS Purine Nucleotide Degradation 2’-deoxyribonucleotides are produced from ribonucleoside Nucleic acids from diet are degraded in the small intestine diphosphates by ribonucleotide reductase →Hydrolyzed to nucleotides by pancreatic nucleases →DNA synthesis requires 2’-deoxyribonucleotides →Further degradation by intestinal enzymes to nucleosides Ribonucleotide reductase reduces: Uric acid is the end product of purine degradation in the →Purine nucleoside diphosphates (ADP and GDP) intestine →Pyrimidine nucleoside diphosphates (CDP and UDP) Purine nucleotides from de novo synthesis are mainly →Their deoxy forms (dADP, dGDP, dCDP, and dUDP). degraded in the liver →The enzyme is composed of two nonidentical subunits: Degradation In the Small Intestine R1 (α) and the smaller R2 (β) Pancreas secretes ribonucleases and →Two sulfhydryl (-SH) groups on the enzyme (R1 subunit) deoxyribonucleases that hydrolyze dietary RNA and DNA act as immediate donors of the H atoms needed to Resulting oligonucleotides are further hydrolyzed by reduce the 2’-hydroxyl groups pancreatic phosphodiesterases, producing 3’- and Forms a disulfide bond 5’-mononucleotides The disulfide bond formed while producing the 2’-deoxy Nucleotidases remove the phosphate groups hydrolytically carbon must be reduced for ribonucleotide reductase to →Nucleosides are released and then degraded by continue its functions nucleosidases to free bases and (deoxy) ribose →Thioredoxin, a protein coenzyme of ribonucleotide 1-phosphate reductase, acts as the source of the reducing Dietary purine bases are degraded to uric acid in equivalents enterocytes The two -SH groups of thioredoxin donate their H →Uric acid enters the blood before excretion in urine atoms to ribonucleotide reductase, forming a disulfide bond in the process Reduced thioredoxin is regenerated to allow the protein coenzyme to continue its normal functions. →NADPH + H+ produce the reducing equivalents →Thioredoxin reductase catalyzes the reaction Figure 22. Conversion of ribonucleotides to deoxyribonucleotides Regulation of Deoxyribonucleotide Synthesis Figure 23. Degradation of Dietary Nucleic Acids The regulation of ribonucleotide reductase is complex. R1 contains two distinct allosteric sites (“activity sites”) Uric Acid Formation involved in regulating enzymatic activity. An amino group is removed from either: dATP binding to allosteric sites on R1 inhibits the overall →AMP to produce IMP (catalyzed by adenylate catalytic activity of the enzyme. deaminase) →Prevents the reduction of any of the four nucleoside →Adenosine to produce inosine (catalyzed by adenosine diphosphates deaminase) →Prevents DNA synthesis IMP and GMP are converted into inosine and guanosine, ATP binding to allosteric sites on R1 activates the overall respectively (catalyzed by 5’-nucleotidase) catalytic activity of the enzyme. →”Conversion into nucleosides” Binding of nucleoside triphosphates to additional allosteric Inosine and guanosine are converted into hypoxanthine sites on R1 (“substrate specificity sites”) regulates and guanine, respectively substrate specificity →“Conversion into purine bases” →Increases the conversion of ribonucleotides to Guanine is deaminated to form xanthine deoxyribonucleotides CELL 02.11 Nitrogen Metabolism and Nucleic Acid Metabolism 12 of 14  Hypoxanthine is oxidized to xanthine (catalyzed by →Step 4: Dihydroorotate is oxidized by dihydroorotate xanthine oxidase) dehydrogenase to produce the completed pyrimidine Xanthine is further oxidized by the same enzyme to uric acid ring orotic acid (orotate) This enzyme is the only one in pyrimidine synthesis Diseases Associated with Purine Degradation that is in the inner mitochondrial membrane; all others Gout are cytosolic →Due to high levels of uric acid in blood (hyperuricemia) Steps in pyrimidine nucleotide synthesis: because of the overproduction (less common) or →Step 1: Orotate is converted to orotidine monophosphate underexcretion (more common) of uric acid (OMP) by orotate phosphoribosyltransferase, releasing Hyperuricemia can cause monosodium urate (MSU) pyrophosphate crystals to be deposited in the joints PRPP is the ribose 5-phosphate donor Crystals may trigger an inflammatory response, Biologically irreversible reaction causing first acute, then chronic gouty arthritis →Step 2: Orotidine monophosphate is decarboxylated by Nodular masses of MSU crystals may be deposited in OMP decarboxylase to form uridine monophosphate soft tissues, causing chronic tophaceous gout (UMP) Uric acid stone formation (urolithiasis) can occur in →Step 3: UMP is sequentially phosphorylated to UDP and kidney UTP Mutations in X-linked PRPP synthetase causes the Phosphoribosyltransferase and decarboxylase are enzyme to have an increased Vmax for PRPP separate catalytic domains of a single polypeptide called production and decreased Km for ribose 5-phosphate, UMP synthase or decreased sensitivity to purine nucleotides Cytidine triphosphate synthesis: ○ Increased PRPP availability increases purine →UTP is aminated by CTP synthetase to form CTP, with production glutamine providing the nitrogen Lesch-Nyhan syndrome can cause hyperuricemia due →Some of the CTP is dephosphorylated to CDP (substrate to decreased salvage of hypoxanthine and guanine, for ribonucleotide reductase) increasing PRPP availability →dCDP product can be phosphorylated to dCTP for DNA Underexcretion of uric acid can be due to inherent synthesis or dephosphorylated to dCMP that is excretory defects, secondary effects of known deaminated to dUMP diseases (ex. Lactic acidosis) and environmental Deoxythymidine monophosphate synthesis exposure such as drug use and lead exposure →Thymidylate synthase uses N5,N10-methylene-THF as Adenosine Deaminase (ADA) Deficiency the source of the methyl group to convert dUMP to →Causes accumulation of adenosine, which is converted deoxythymidine monophosphate (dTMP) to its ribonucleotide or deoxyribonucleotide forms by Thymine analogs such as 5-fluorouracil inhibit kinases thymidylate synthase →Elevated dATP levels inhibit ribonucleotide reductase, Pyrimidine salvage and degradation thus inhibiting production of all deoxyribose-containing →The pyrimidine ring can be opened and degraded to nucleotides highly soluble products: →Cells cannot synthesize DNA and divide β-alanine (from degradation of CMP and UMP) Can lead to developmental arrest and apoptosis β-aminoisobutyrate (from TMP degradation) →Severe form causes severe combined immunodeficiency ammonia disease, decreasing T cells, B cells, and natural killer CO2 cells →Pyrimidine bases can be salvaged to nucleosides, which are phosphorylated to nucleotide Pyrimidine Synthesis and Degradation Pyrimidine ring is synthesized before attachment to a ribose 5-phosphate donated by PRPP Atoms in the ring are donated by glutamine, CO2, and aspartate First 3 enzymes in the pathway are catalytic domains of a single polypeptide called CAD Steps in pyrimidine synthesis: →Step 1: Carbamoyl phosphate is synthesized from glutamine and CO2, catalyzed by carbamoyl phosphate synthetase II (CPS II) Regulated step of the pathway CPS II is inhibited by uridine triphosphate (end-product of the pathway) and activated by PRPP →Step 2: Aspartate transcarbamoylase catalyzes formation of carbamoyl aspartate →Step 3: Dihydroorotase closes the pyrimidine ring, forming dihydroorotate CELL 02.11 Nitrogen Metabolism and Nucleic Acid Metabolism 13 of 14  FREEDOM SPACE “I won’t read this trans – it’s too long.” “Identify the structure colored purple.” CELL 02.11 Nitrogen Metabolism and Nucleic Acid Metabolism 14 of 14